Surface translocation of ACE2 and TMPRSS2 upon TLR4/7/8 activation is required for SARS-CoV-2 infection in circulating monocytes

Infection of human peripheral blood cells by SARS-CoV-2 has been debated because immune cells lack mRNA expression of both angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease type 2 (TMPRSS2). Herein we demonstrate that resting primary monocytes harbor abundant cytoplasmic ACE2 and TMPRSS2 protein and that circulating exosomes contain significant ACE2 protein. Upon ex vivo TLR4/7/8 stimulation, cytoplasmic ACE2 was quickly translocated to the monocyte cell surface independently of ACE2 transcription, while TMPRSS2 surface translocation occurred in conjunction with elevated mRNA expression. The rapid translocation of ACE2 to the monocyte cell surface was blocked by the endosomal trafficking inhibitor endosidin 2, suggesting that endosomal ACE2 could be derived from circulating ACE2-containing exosomes. TLR-stimulated monocytes concurrently expressing ACE2 and TMPRSS2 on the cell surface were efficiently infected by SARS-CoV-2, which was significantly mitigated by remdesivir, TMPRSS2 inhibitor camostat, and anti-ACE2 antibody. Mass cytometry showed that ACE2 surface translocation in peripheral myeloid cells from patients with severe COVID-19 correlated with its hyperactivation and PD-L1 expression. Collectively, TLR4/7/8-induced ACE2 translocation with TMPRSS2 expression makes circulating monocytes permissive to SARS-CoV-2 infection.


mRNA expression of ACE2 and TMPRSS2 in monocytes and T cells upon ex vivo stimulation.
(a) PBMC samples were ex vivo cultured in the presence or absence of R848 or LPS for 4 h and stained with anti-CD14 and anti-ACE2 antibodies on cell surface. ACE2 + CD14 + , ACE2 -CD14 + and total monocytes (MO) were sorted for qPCR analysis. Relative expression of ACE2 mRNA in the indicted monocyte populations compared to that in human prostate adenocarcinoma LNCaP cell line. N = 3-4 biologically independent samples per group. (b, c) PBMCs were ex vivo cultured with PMA plus ionomycin for 4 h. ACE2 + CD3 + T, ACE2 -CD3 + T and total CD3 + T cells were sorted for qPCR analysis. (b) Relative expression of ACE2 mRNA in the indicted T cell populations compared to that in LNCaP cells. N = 3 biologically independent samples per group. (c) Relative expression of TMPRSS2 mRNA in the indicated populations compared to that in the total T cells without treatment. N = 3 biologically independent samples per group.  (a) PBMCs were cultured ex vivo in the presence of PMA plus ionomycin (P/I) for 4 h, followed by flow cytometry analysis of surface ACE2 in total T cells and CD4 + and CD8 + T cells. (b) The frequencies of the indicated T cell populations expressing surface ACE2 as in a (n = 3 biologically independent samples/group).
Supplementary Figure S4 ACE2 protein levels in PBMCs and monocytes upon ex vivo stimulation with R848.
PBMCs isolated from healthy donors were ex vivo cultured in the presence or absence of R848 for 24 h followed by CD14 + monocyte (MO) enrichment (>85% purity). A total of 60 µg protein from untreated (Unt) or R848-treated PBMCs and enriched monocytes were loaded to the gels for immunoblotting. Representative blot bands showing the expression of ACE2. GAPDH serves as a loading control. Huh-7 and Vero cell lines served as positive controls.   (a) Imaging flow cytometry analysis of cytoplasmic and surface ACE2 protein in CD14 + monocytes treated (Tx) with or without LPS up to 4 h. Each cell is represented by the row of images that include bright field (BF), DAPI (purple), CD14 (turquoise), ACE2 (red), and the overlapping image merged with DAPI, CD14, and ACE2. Histograms of ACE2 intensity on the cell surface of CD14 + monocytes treated with LPS (b). Grey line represents the isotype control group (Iso). The column charts of mean fluorescence intensity (MFI) of surface ACE2 at other indicated conditions are shown on the right (n = 3 biologically independent samples/group).  Imaging flow cytometry of intracellular and surface ACE2 in peripheral blood monocytes.

Supplementary Figure S5
PBMCs were treated (Tx) with or without R848 or LPS up to 4 h and analyzed for ACE2 expression on the cell surface or intracellularly (ICS) in CD14 + monocytes. Each cell is represented by the row of images that include bright field (BF), DAPI (purple), CD14 (turquoise), ACE2 (red), and the overlapping image merged with DAPI, CD14, and ACE2. Four cells are shown for each condition.
Supplementary Figure S7 ACE2 transcriptomic analysis of CD45 + haematopoietic cells in tissues from human embryos.
PBMCs were cultured in the presence of R848 or LPS or medium alone for 2 h prior to infection with SARS-CoV-2 at MOI = 1 or 3 for 24 h. Flow cytometry analysis of the surface ACE2 and intracellular SARS-CoV-2 nucleocapsid (N) protein in cDC from mock-infected and virus-infected cells. Data were collected from a pool of PBMCs from 3 healthy control samples. Two independent infection experiments were performed with similar results.   Supplementary Figure S12 CyTOF gating strategy of myeloid populations in whole blood upon ex vivo stimulation. Whole blood samples were ex vivo stimulated in the presence or absence of LPS for 4 h, and then stained with metal-conjugated antibodies for mass cytometry analysis. Violin plots showing the frequencies of myeloid compartment expressing surface ACE2 (a) and PD-L1 (b) from untreated samples (n = 7 healthy controls (HC); n = 7 severe COVID-19) as well as LPS-treated samples (n = 7 HC; n = 15 moderate COVID-19; and n = 16 severe COVID-19) of cohort.
Supplementary Figure S13 a b % of max events  Co-expression of ACE2 and CD16 in CD14 + monocytes upon R848 or LPS ex vivo stimulation.
Whole blood samples were ex vivo stimulated in the presence or absence of R848 or LPS for 4 h, and then stained with metal-conjugated antibodies for mass cytometry analysis. (a) Mass cytometry analysis of ACE2 and CD16 expression on the cell surface of CD14 + monocytes from R848 or LPStreated (n = 7 HC; n = 14 moderate COVID-19; and n = 15 severe COVID-19) samples. (b) Violin plots of the frequencies of ACE2 + CD16 + cells within CD14 + monocytes as shown in (a).
Supplementary Figure S15